Albumin Oxidation and Albumin Glycation Discordance During Type 2 Diabetes Therapy: Biological and Clinical Implications.

Aims: Cys34 albumin redox modifications (reversible "cysteinylation" and irreversible "di/trioxidation"), besides being just oxidative stress biomarkers, may have primary pathogenetic roles to initiate and/or aggravate cell, tissue, and vascular damage in diabetes. In an exploratory "proof-of-concept" pilot study, we examined longitudinal changes in albumin oxidation during diabetes therapy. Methods: Mass spectrometric analysis was utilized to monitor changes in human serum albumin (HSA) post-translational modifications {glycation [glycated albumin (GA)], cysteinylation [cysteinylated albumin (CA) or human non-mercaptalbumin-1; reversible], di/trioxidation (di/trioxidized albumin or human non-mercaptalbumin-2; irreversible), and truncation (truncated albumin)} during ongoing therapy. Four informative groups of subjects were evaluated [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity, and healthy controls] at baseline, and subjects with diabetes were followed for a period up to 280 days. Results: At baseline, T2DM was associated with relatively enhanced albumin cysteinylation (CA% total) compared with T1DM (P = 0.004), despite comparable mean hyperglycemia (P values: hemoglobin A1c = 0.09; GA = 0.09). T2DM, compared with T1DM, exhibited selectively and significantly higher elevations of all the "individual" glycated cum cysteinylated ("multimodified") albumin isoforms (P values: CysHSA+1G = 0.003; CysHSA+2G = 0.007; and CysHSA+3G = 0.001). Improvements in glycemic control and decreases in albumin glycation during diabetes therapy in T2DM were not always associated with concurrent reductions of albumin cysteinylation, and in some therapeutic situations, albumin cysteinylation worsened (glycation-cysteinylation discordance). Important differences were observed between the effects of sulfonylureas and metformin on albumin molecular modifications. Conclusions: T2DM was associated with higher oxidative (cysteinylation) and combined (cysteinylation plus glycation) albumin molecular modifications, which are not ameliorated by improved glucose control alone. Further studies are required to establish the clinical significance and optimal therapeutic strategies to address oxidative protein damage and resulting consequences in diabetes.

William L. Mock and Cucurbituril: The Resurrection of the Behrend Paper of 1905.

In 1905, Robert Behrend reported the preparation of a substance that remained a dry powder despite absorbing water and that had the ability to absorb dyes from solution. Over seven decades later, William L. Mock repeated the Behrend synthesis and discovered cucurbituril, a remarkably symmetric molecule with a central cavity welcoming diverse guests. Mock et al. ploughed a lonely furrow in the 1980s, establishing the versatility of cucurbituril in the field of supramolecular chemistry, including a demonstration of the Huisgen click reaction in a molecular cavity.

Cysteine-free cone snail venom peptides: Classification of precursor proteins and identification of mature peptides.

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.

Differential Spectrum of Albumin Glycation, Oxidation, and Truncation in Type 2 and Type 1 Diabetes: Clinical and Biological Implications.

Background: Albumin, the most abundant and physiologically vital serum protein, accumulates a range of chemical modifications, as consequence of encounters with large number of reactive molecules whose concentrations increase in serum under pathological conditions. Methods: In a "proof of concept" study, mass spectrometric analysis was utilized to quantitate albumin post-translational modifications (glycation, oxidation, and truncation; individual isoforms and total) in four informative subject groups [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity and healthy; all with estimated glomerular filtration rate ≥60 mL/(min·m2)]. Besides glycated albumin (GA/mass spectrometry), glycated serum protein (GSP/nitro blue tetrazolium colorimetry), and glycated hemoglobin (HbA1c/high-performance liquid chromatography) were also measured. Results: A wide spectrum of albumin molecular modifications was identified in diabetes, with significant differences between T2DM and T1DM. Albumin glycation: GA correlated more strongly with HbA1c in T1DM, compared to T2DM. Higher albumin glycation isoforms (human serum albumin +3G/2G) were more stable and discriminative markers of mean glycemia. Albumin oxidation: T2DM, in comparison with T1DM, showed enhanced oxidative and dual (glycation plus oxidation) modifications, representing extreme molecular pathology. Albumin truncation: There was dramatic reduction ("deficiency") of truncated albumin isoforms in T2DM, and significant reduction in T1DM. Albumin truncation negatively correlated with severity of albumin glycation (mean glycemia) and albumin oxidation (cysteinylation). Possible mechanisms of insulin resistance, with associated increased free fatty acids binding to albumin, in stimulating albumin oxidation and inhibiting albumin glycation ("metabolic cross talks") are reviewed. Conclusions: Albumin molecular modifications in diabetes, together with significant differences between T2DM and T1DM, suggest possible role for insulin resistance in their genesis and consequent cell, tissue, and vascular dysfunction/damage. Albumin molecular fingerprinting can provide valuable insights into pathogenesis, diagnosis, monitoring, and future therapies for diabetes. Identification of biomarker battery ("albuminomics," "diabetomics") driven diverse "healthy," prediabetes, obesity, and T2DM phenotypes represents additional novel step toward precision medicine in diabetes and related disorders.

Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin.

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol-1, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apo-protein has been determined, revealing that large intersubunit rearrangements occur across apo- and holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis, and computational studies, including density functional theory (DFT) and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition-state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site.

Mechanistic Investigations on N-Cα Bond Cleavages in Dibasic Peptides Containing Internal Lys and Arg Residues.

The model nonapeptide AAARAAKAG* (* indicates amide) is used to explore N-Cα bond fragmentation under CID-MS conditions. Neighboring group participation and the effect of positioning of Lys and Arg residues on N-Cα bond cleavage is established using a library of synthetic peptide analogues. The importance of the Arg residue at position 4 and the i to i+3 spacing between Arg and Lys residues in determining the formation of the N-Cα bond cleaved product ions (cn) is demonstrated by a comparative MS study of positional variants in analogue peptides. The effect of shortening of the Lys side chain has been established using ornithine (Orn) and diaminobutyric acid (Dab) analogues. The involvement of the Lys residue in mediating the N-Cα bond cleavage is further probed using Nε-dimethyl and isotopically labeled 15Nα, Îµ lysine residues. MSn experiments reveal that the c6 ion originates from a doubly charged dehydrated b8 ion [b8-18]2+. The mechanism of this unusual fragmentation process has been probed by using position 8 analogues (Gly, Ala, and Aib). A plausible mechanism is proposed for the origin of the c6 ion, which involves C-terminus lactam formation followed by transannular cyclization and dehydration. The results presented in this study highlight the role of reactive side chain functionalities in promoting noncanonical fragmentation pathways.

Mass Spectrometric Analysis of Cyclotides from Clitoria ternatea: Xxx-Pro Bond Fragmentation as Convenient Diagnostic of Pro Residue Positioning.

Cyclotides, a class of macrocyclic plant peptides, characterized by a cyclic backbone and three inter-locking disulfide bonds, may be divided into two major structural subfamilies, Möbius and Bracelet, based on the presence or absence of a specific proline residue. The present study describes the suite of cyclotides obtained from Clitoria ternatea, characterized by LC-MS and MS/MS techniques. Notable variations in product ion distributions were observed in cyclotides belonging to different structural subfamilies based on the number and positions of proline residues. For instance, Cter M which is an abundant Möbius cyclotide in this plant containing three proline residues, displayed distinct b- and y- ion characteristics in the MS/MS spectra compared to Cliotide T1, another commonly identified cyclotide but belonging to the Bracelet subfamily having two proline residues. The distinct fragmentation pattern of prototypical cyclotides of each structural subfamily, determined by Xxx-Pro bond fragmentation, was used to rapidly identify and sequence a novel cyclotide ctr pep 30 from this plant.

Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation.

Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or ß-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π∗ interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.

Transcriptomic profiling of the medicinal plant Clitoria ternatea: identification of potential genes in cyclotide biosynthesis.

Clitoria ternatea a perennial climber of the Fabaceae family, is well known for its agricultural and medical applications. It is also currently the only known member of the Fabaceae family that produces abundant amounts of the ultra-stable macrocyclic peptides, cyclotides, across all tissues. Cyclotides are a class of gene-encoded, disulphide-rich, macrocyclic peptides (26-37 residues) acting as defensive metabolites in several plant species. Previous transcriptomic studies have demonstrated the genetic origin of cyclotides from the Fabaceae plant family to be embedded in the albumin-1 genes, unlike its counterparts in other plant families. However, the complete mechanism of its biosynthesis and the repertoire of enzymes involved in cyclotide folding and processing remains to be understood. In this study, using RNA-Seq data and de novo transcriptome assembly of Clitoria ternatea, we have identified 71 precursor genes of cyclotides. Out of 71 unique cyclotide precursor genes obtained, 51 sequences display unique cyclotide domains, of which 26 are novel cyclotide sequences, arising from four individual tissues. MALDI-TOF mass spectrometry analysis of fractions from different tissue extracts, coupled with precursor protein sequences obtained from transcriptomic data, established the cyclotide diversity in this plant species. Special focus in this study has also been on identifying possible enzymes responsible for proper folding and processing of cyclotides in the cell. Transcriptomic mining for oxidative folding enzymes such as protein-disulphide isomerases (PDI), ER oxidoreductin-1 (ERO1) and peptidylprolyl cis-trans isomerases (PPIases)/cyclophilins, and their levels of expression are also reported. In particular, it was observed that the CtPDI genes formed plant-specific clusters among PDI genes as compared to those from other plant species. Collectively, this work provides insights into the biogenesis of the medicinally important cyclotides and establishes the expression of certain key enzymes participating in peptide biosynthesis. Also, several novel cyclotide sequences are reported and precursor sequences are analysed in detail. In the absence of a published reference genome, a comprehensive transcriptomics approach was adopted to provide an overview of diverse properties and constituents of C. ternatea.

Mechanistic Insights into an Unusual Side-Chain-Mediated N-Cα Bond Cleavage under Collision-Induced Dissociation Conditions in the Disulfide-Containing Peptide Conopressin.

Conopressin, a nonapeptide disulfide CFIRNCPKG amide present in cone snail venom, undergoes a facile cleavage at the Cys6-Pro7 peptide bond to yield a disulfide bridged b6 ion. Analysis of the mass spectral fragmentation pattern reveals the presence of a major fragment ion, which is unambiguously assigned as the tripeptide sequence IRN amide. The sequence dependence of this unusual fragmentation process has been investigated by comparing it with the fragmentation patterns of related peptides, oxytocin (CYIQNCPLG amide), Lys-vasopressin (CYFQNCPKG amide), and a series of synthetic analogues. The results establish the role of the Arg4 residue in facilitating the unusual N-Cα bond cleavage at Cys6. Structures are proposed for a modified disulfide bridged fragment containing the Cys1 and Cys6 residues. Gas-phase molecular dynamics simulations provide evidence for the occurrence of conformational states that permit close approach of the Arg4 side chain to the Cys6 Cß methylene protons.

Pregabalin peptides: conformational comparison of γ3- and γ4-substituted γ-amino acids in αγααα pentapeptides.

Gamma-aminobutyric acid (GABA, gammaAbu), an unsubstituted gamma-amino acid, is an important inhibitory neurotransmitter in the mammalian brain. The role of GABA in the treatment of epilepsy has triggered a great deal of interest in substituted gamma-amino acids, which may serve as GABA analogs, acting as inhibitors of GABA aminotransferase. Pregabalin (Pgn), a well-known antiepileptic drug, is also a beta-substituted gamma3-amino acid. Pregabalin and gamma4Leu, an isomer of the pregabalin (Pgn) residue, both carrying the same isobutyryl group in the side chain, were introduced in the present study to have a comparison of their respective conformational differences as well as their role in influencing the overall conformation of the peptides, they are inserted in. Two alpha-gamma-alpha-alpha-alpha hybrid pentapeptides were designed that contain Aib-Pgn and Aib-gamma4Leu segments at the N terminus. The study provides a detailed analysis of the conformational properties and non-covalent interactions observed in the crystal structures of two polymorphs of the pentapeptide monohydrate, Boc-Aib-(S)Pgn-Leu-Phe-Val-OMe (C38H63N5O8·H2O) and the isomeric pentapeptide, Boc-Aib-gamma4(R)Leu-Leu-Phe-Val-OMe (C38H63N5O8), obtained from single crystal X-ray diffraction experiments.

Cone Snail Glutaminyl Cyclase Sequences from Transcriptomic Analysis and Mass Spectrometric Characterization of Two Pyroglutamyl Conotoxins.

The post-translational modification of N-terminal glutamine (Q) to a pyroglutamyl (Z) residue is observed in the conotoxins produced by marine cone snails. This conversion requires the action of the enzyme glutaminyl cyclase (QC). Four complete QC sequences from the species C. araneosus, C. frigidus, C. litteratus, and C. monile and two partial sequences from C. amadis and C. miles have been obtained by analysis of transcriptomic data. Comparisons with mammalian enzyme sequences establish a high level of identity and complete conservation of functional active site residues, including a cluster of hydrogen-bonded acidic side chains. Mass spectrometric analysis of crude venom samples coupled to conotoxin precursor protein sequences obtained from transcriptomic data establishes the presence of pyroglutamyl conotoxins in the venom of C. frigidus and C. amadis. The C. frigidus peptide belongs to the M superfamily, with cysteine framework III, whereas the C. amadis peptide belongs to the divergent superfamily with cysteine framework VI/VII. Additionally, gamma carboxylation of glutamic acid and hydroxylation of proline are observed in the C. frigidus peptide. Mass spectral data are available via ProteomeXchange with identifier PXD009006.

Contryphan Genes and Mature Peptides in the Venom of Nine Cone Snail Species by Transcriptomic and Mass Spectrometric Analysis.

The occurrence of contryphans, a class of single-disulfide-bond-containing peptides, is demonstrated by the analysis of the venom of nine species of cone snails. Ten full gene sequences and two partial gene sequences coding for contryphan precursor proteins have been identified by next-generation sequencing and compared with available sequences. The occurrence of mature peptides in isolated venom has been demonstrated by LC-ESI-MS/MS analysis. De novo sequencing of reduced, alkylated contryphans from C. frigidus and C. araneosus provides evidence of sequence variation and post-translational modification, notably gamma carboxylation of glutamic acid. The characterization of Fr965 (C. frigidus) provides a rare example of a sequence lacking Pro at position 5 in the disulfide loop. The widespread occurrence of contryphan genes and mature peptides in the venom of diverse cone snails is suggestive of their potential biological significance.

Intramolecular backbone···backbone hydrogen bonds in polypeptide conformations. The other way around: ɛ-turn.

In this study, we performed a detailed literature survey of the ɛ-turn in peptides and proteins. This three-dimensional structural feature is characterized by an eleven-membered pseudo-cycle closed by an intramolecular backbone…backbone H-bond. Interestingly, in this motif the direction of the N-H…O = C H-bond runs opposite to that of the much more popular and extensively investigated α-, ß-, and γ-turns. We did not authenticate unequivocally the ɛ-turn main-chain reversal topology in any linear short peptide. However, it is frequently observed in small cyclic peptides formed by four, five, and six amino acid residues with stringent geometric requirements. Rather surprisingly, ɛ-turns do occur in proteins, although to a relatively moderate extent, as an isolated feature or in the turn segment of hairpin motifs based on two antiparallel, pleated ß-strands. Moreover, the ɛ-turn may also host not only the seven-membered, intramolecularly H-bonded, pseudo-cycle termed γ-turn, either of the classic or inverse type, but also one (or even two) cis peptide bond(s) or a ß-bulge conformation. Based on their ϕ, ψ backbone torsion angles, we were able to classify the protein ɛ-turns in six different families. Conformational energy computations using the DFT methodology were also performed on the ɛ-turns adopted by the amino acid triplet -Gly-Gly-Gly- (Gly is the most commonly found residue at each of the three positions in our analysis of proteins). Again, in this computational study, six families of turns were identified, but only some of them resemble rather closely those extracted from our investigation on proteins.

Conformational properties and aggregation of homo-oligomeric ß3 (R)-valine peptides in organic solvents.

The conformational characteristics of protected homo-oligomeric Boc-[ß3 (R)Val]n -OMe, n = 1, 2, 3, 4, 6, 9, and 12 have been investigated in organic solvents using nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) absorption spectroscopy and circular dichroism (CD) methods. The detailed 1 H NMR analysis of Boc-[ß3 (R)Val]12 -OMe reveals that the peptide aggregates extensively in CDCl3 , but is disaggregated in 20%, (v/v) dimethyl sulfoxide (DMSO) in CDCl3 and in CD3 OH. Limited assignment of the N-terminus NH groups, together with solvent dependence of NH chemical shifts and temperature coefficients provides evidence for 14-helix conformation in the 12-residue peptide. FTIR analysis in CHCl3 establishes that the onset of folding and aggregation, as evidenced by NH stretching bands at 3375 cm-1 (intramolecular) and 3285 cm-1 (intermolecular), begins at the level of the tetrapeptide. The observed CD bands, 214 nm (negative) and 198 nm (positive), support 14-helix formation in the 9 and 12 residue sequences. The folding and aggregation tendencies of homo-oligomeric α-, ß-, and γ- residues is compared in the model peptides Boc-[ωVal]n -NHMe, ω = α, ß, and γ and n = 1, 2, and 3. Analysis of the FTIR spectra in CHCl3 , establish that the tendency to aggregate at the di and tripeptide level follows the order ß > α∼γ, while the tendency to fold follows the order γ > ß > α.

Homooligomeric ß3 (R)-valine peptides: Transformation between C14 and C12 helical structures induced by a guest Aib residue.

Novel helical, structures unprecedented in the chemistry of α-polypeptides, may be found in polypeptides containing ß and γ amino acids. The structural characterization of C12 and C14 -helices in oligo ß-peptides was originally achieved using conformationally constrained cyclic ß-residues. This study explores the conformational characteristics of proteinogenic ß3 residues in homooligomeric sequences and addresses the issue of inducing a transition between C14 and C12 helices by the introduction of a guest α-residue. Folded C14 -helical structures are demonstrated for the nonapeptide Boc-[ß3 (R)Val]9 -OMe by NMR methods in CDCl3 -DMSO mixtures, while the peptide was found to be aggregated in CDCl3 . The insertion of a guest Aib residue into an oligo-ß-valine sequence in the octapeptide model Boc-[(ß3 (R)Val)3 -Aib-(ß3 (R)Val]4 -OMe results in well dispersed NH region in the NMR spectrum indicating folded structures in CDCl3 . Structure calculations for both the peptides using NOE distance constraints support a C14 helical structure in the homooligomer which transform into a C12 helix on introduction of the guest Aib residue.

Unexpected functional implication of a stable succinimide in the structural stability of Methanocaldococcus jannaschii glutaminase.

Protein ageing is often mediated by the formation of succinimide intermediates. These short-lived intermediates derive from asparaginyl deamidation and aspartyl dehydration and are rapidly converted into ß-aspartyl or D-aspartyl residues. Here we report the presence of a highly stable succinimide intermediate in the glutaminase subunit of GMP synthetase from the hyperthermophile Methanocaldoccocus jannaschii. By comparing the biophysical properties of the wild-type protein and of several mutants, we show that the presence of succinimide increases the structural stability of the glutaminase subunit. The protein bearing this modification in fact remains folded at 100 °C and in 8 M guanidinium chloride. Mutation of the residue following the reactive asparagine provides insight into the factors that contribute to the hydrolytic stability of the succinimide. Our findings suggest that sequences that stabilize succinimides from hydrolysis may be evolutionarily selected to confer extreme thermal stability.

Crystallographic characterization of the α,γ C12 helix in hybrid peptide sequences.

The solid-state conformations of two αγ hybrid peptides Boc-[Aib-γ(4) (R)Ile]4 -OMe 1 and Boc-[Aib-γ(4) (R)Ile]5 -OMe 2 are described. Peptides 1 and 2 adopt C12 -helical conformations in crystals. The structure of octapeptide 1 is stabilized by six intramolecular 4 → 1 hydrogen bonds, forming 12 atom C12 motifs. The structure of peptide 2 reveals the formation of eight successive C12 hydrogen-bonded turns. Average backbone dihedral angles for αγ C12 helices are peptide 1, Aib; φ (°) = -57.2 ± 0.8, ψ (°) = -44.5 ± 4.7; γ(4) (R)Ile; φ (°) = -127.3 ± 7.3, θ1 (°) = 58.5 ± 12.1, θ2 (°) = 67.6 ± 10.1, ψ (°) = -126.2 ± 16.1; peptide 2, Aib; φ (°) = -58.8 ± 5.1, ψ (°) = -40.3 ± 5.5; ψ(4) (R)Ile; φ (°) = -123.9 ± 2.7, θ1 (°) = 53.3 θ 4.9, θ 2 (°) = 61.2 ± 1.6, ψ (°) = -121.8 ± 5.1. The tendency of γ(4) -substituted residues to adopt gauche-gauche conformations about the C(α) -C(ß) and C(ß) -C(γ) bonds facilitates helical folding. The αγ C12 helix is a backbone expanded analog of α peptide 310 helix. The hydrogen bond parameters for α peptide 310 and α-helices are compared with those for αγ hybrid C12 helix. Copyright © 2016 European Peptide Society and John Wiley & Sons.

Mass spectrometric analysis of dimer-disrupting mutations in Plasmodium triosephosphate isomerase.

Electrospray ionization mass spectrometry (ESI MS) under nanospray conditions has been used to examine the effects of mutation at two key dimer interface residues, Gln (Q) 64 and Thr (T) 75, in Plasmodium falciparum triosephosphate isomerase. Both residues participate in an intricate network of intra- and intersubunit hydrogen bonds. The gas phase distributions of dimeric and monomeric protein species have been examined for the wild type enzyme (TWT) and three mutants, Q64N, Q64E, and T75S, under a wide range of collision energies (40-160 eV). The results established the order of dimer stability as TWT > T75S > Q64E âˆ¼ Q64N. The mutational effects on dimer stability are in good agreement with the previously reported estimates, based on the concentration dependence of enzyme activity. Additional experiments in solution, using inhibition of activity by a synthetic dimer interface peptide, further support the broad agreement between gas phase and solution studies.